DGCR2-Targeting Affibody Molecules for Beta-Cell Drug Delivery and Imaging Reagent Development

A common pathological feature of both type 1 and type 2 diabetes mellitus is the progressive loss of insulin-secreting β-cells in the pancreas. Novel approaches that enable direct and specific targeting of β-cells offer a promising platform for the delivery of pharmaceutical agents. Positron emission tomography (PET) and other imaging modalities rely on the efficient and specific delivery of imaging probes, which can deepen our understanding of diabetes etiology and provide biomarkers for viable β-cell mass in the pancreas and islet grafts. Previously, the DiGeorge syndrome critical region gene 2 (DGCR2) protein was identified as a β-cell-specific protein in the pancreas; however, high-affinity binders suitable for targeted drug delivery or molecular imaging have remained lacking. Affibody molecules are small scaffold affinity proteins with excellent molecular imaging properties. Cheung et al. ^[1]further confirmed the presence of DGCR2 in both pancreatic and stem cell (SC)-derived β-cells, and described the construction and screening of multiple Affibody molecule candidates targeting human DGCR2. Through an in-house directed evolution approach, the study generated and evaluated novel DGCR2-binding Affibody variants for thermal stability and binding affinity. These variants were subsequently developed as carriers for targeted delivery of imaging reagents to β-cells. The Affibody molecule ZDGCR2:AM106 demonstrated nanomolar affinity, favorable stability, and biodistribution profiles, with minimal cytotoxicity to islet cells, positioning it as a lead candidate for further development of specific delivery of drugs and imaging agents to β-cells, while also offering new perspectives for diagnostic and therapeutic targeted drug development in various diseases.

1.Principle of Affibody-Mediated Targeted Delivery:

图1 利用DGCR2靶向递送药物和成像试剂至β细胞的概念 Afffbody结构特征：.png

Figure 1. Concept of DGCR2-targeted delivery of drugs and imaging reagents to β-cells.

2.Structural Features of Affibody Molecules:

Affibody molecules are small scaffold proteins of approximately 7 kDa (58 amino acids). They are composed of three α-helices, with helices 1 and 2 engineered to provide a binding surface through the incorporation of 13–15 variable amino acid residues, while the remaining sequence, including helix 3, remains constant. Affibody molecules have been widely used as tracers for molecular imaging in both preclinical and clinical studies, demonstrating favorable imaging contrast, largely attributable to their small size and generally high affinity and specificity. Due to their small dimensions, these molecules undergo rapid renal clearance, conferring favorable pharmacokinetic properties and excellent contrast for molecular imaging. Furthermore, Affibody molecules extravasate rapidly from the bloodstream and exhibit efficient tissue penetration, which facilitates targeting of pathological tissues.

图2 Afffbody结构图.png

Figure 2. Structural diagram of Affibody molecule.

3.Affinity Maturation of Affibody Molecules:

To obtain Affibody molecules with enhanced binding capacity for DGCR2, the study designed an affinity maturation library by randomizing 13 positions on the binding surfaces of helices 1 and 2. This DNA library was cloned into an E. coli display vector, and the plasmid library was introduced into cells via electroporation to obtain transformants. After validation, the library was expressed on the E. coli surface and subjected to four rounds of FACS sorting and analysis, with alternating cell growth and amplification between each round. Flow cytometric visualization of target-binding characteristics within the library revealed progressive enrichment of DGCR2-positive clones across successive sorting rounds (Figure 3).

图3 大肠杆菌展示Affibody成熟文库在四个连续FACS轮次中的结果图.png

Figure 3. Results of E. coli-displayed Affibody maturation library across four consecutive FACS rounds.

4.Thermal Stability Characterization of Affibody Variants:

The newly selected variants for further characterization (ZDGCR2:AM106, ZDGCR2:AM115, and ZDGCR2:AM156), along with the original Affibody ZDGCR2:3D1, were subjected to site-directed mutagenesis introducing scaffold mutations S42A, E43N, S46A, and S54A. Circular dichroism (CD) spectroscopy performed before and after thermally induced denaturation demonstrated that three of the variants (ZDGCR2:3D1, ZDGCR2:AM106, and ZDGCR2:AM156) fully regained their folded state, indicating excellent thermal stability. All evaluated Affibody molecules exhibited favorable stability at approximately 60 °C (Figure 4).

图4 Affibody在20至100°C范围内通过变温测量评估热稳定性.png

Figure 4. Thermal stability assessment of Affibody variants via variable-temperature measurements from 20 to 100 °C.

5.Affinity Characterization of Affibody Variants (SPR-Based):

Surface plasmon resonance (SPR) biosensor analysis was employed to characterize the interactions between Affibody variants (ZDGCR2:3D1, ZDGCR2:AM106, ZDGCR2:AM115, and ZDGCR2:AM156) and DGCR2. The results demonstrated that all three affinity-matured variants exhibited significantly enhanced binding affinity to DGCR2 compared to the original ZDGCR2:3D1 variant (Figure 5).

图5 Affibody分子SPR亲和力检测结果.png

Figure 5. SPR affinity measurement results for Affibody variants.

6.Functional and Biodistribution Assessment of Indium-111-Labeled DOTA-Conjugated Affibody Variants:

Using SPECT imaging, the biodistribution, background tissue uptake, and clearance of indium-111-labeled Affibody variants were evaluated in the heart, liver, kidneys, spleen, and lungs of rats. All Affibody variants exhibited predominant renal clearance (Figure 6A). Dynamic PET/CT imaging was performed in rats to assess the in vivo biodistribution of [??Ga]ZDGCR2:AM106, with ex vivo organ measurements using a gamma counter for further validation. The results confirmed that [??Ga]ZDGCR2:AM106 was primarily cleared via the kidneys (Figure 6B–D).

图7 体外Affibody暴露对人分离原代胰岛的影响验证.png

Figure 6. Functional and biodistribution validation of indium-111-labeled DOTA-conjugated Affibody variants.

7.Effects of In Vitro Affibody Exposure on Isolated Primary Human Islets

Given that the functional role of DGCR2 in human β-cells remains unclear, it was important to assess whether exposure to ZDGCR2:AM106 adversely affects islet function, insulin secretion, or cell viability. Islet perfusion experiments (n = 3) demonstrated that under high-glucose stimulation, insulin release exhibited a typical biphasic pattern regardless of exposure to high-dose ZDGCR2:AM106 (Figure 7A). Thus, exposure of human islets to ZDGCR2:AM106 did not inhibit or enhance glucose-stimulated insulin secretion, which could otherwise interfere with drugs intended to modulate insulin secretion or cause adverse effects such as hypoglycemia. Furthermore, incubation of human islets with ZDGCR2:AM106 at concentrations up to 60 μM did not produce any apparent effects on islet number or viability (Figure 7B).

图7 体外Affibody暴露对人分离原代胰岛的影响验证.png

Figure 7. In vitro effects of Affibody exposure on isolated primary human islets.

8.Summary:

In this study, leveraging the Affibody scaffold technology platform, the authors successfully synthesized and characterized multiple high-affinity binders targeting the β-cell-specific protein DGCR2. Among these, ZDGCR2:AM106 exhibited excellent affinity, stability, refolding capacity, favorable biodistribution, and safety profiles, demonstrating its potential as a lead candidate for further development of DGCR2-targeted drug delivery or imaging reagents.

Tek Biotech (Tianjin) Co., Ltd. has established a comprehensive Affibody development service platform based on phage display and yeast display technologies. We offer one-stop services including affinity maturation library construction, screening, downstream synthesis, and activity validation (covering molecular-level affinity verification, cell-based assays, and animal model studies), providing robust technical support for our clients' research projects.

Reference:

[1] Cheung, P., Persson, J., Zhang, B. et al. DGCR2 targeting affibody molecules for delivery of drugs and imaging reagents to human beta cells. Sci Rep 15, 417 (2025).

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